Apoptosis, or programmed cell death, is a process that multicellular organisms utilize to eliminate unwanted or damaged cells to maintain tissue homeostasis. It plays a critical role in development, immune responses and many other physiological events. Deregulation of apoptosis can lead to diseases such as cancer, immune disorders, and neurodegenerative diseases. Molecularly, apoptosis is executed by proteases known as caspases, and in mammals, a major caspase activation pathway is the cytochrome c-mediated pathway. In this pathway, caspase activation is initiated by cytochrome c release from mitochondria and subsequent assembly of a multimeric protein complex, the apoptosome, which is the central caspase activation machinery. Recently, we identified a new regulatory pathway that controls apoptosome activity. This regulatory pathway can be triggered by a synthetic compound, alpha-(trichloromethyl)-4-Pyridineethanol (PETCM), and the pathway is modulated by both the tumor suppressor PHAP (putative HLA-associated protein) and the oncoprotein prothymosin-alpha (ProT), indicating its potential involvement in oncogenesis. In this proposal, we will investigate the molecular mechanisms of the PETCM-initiated, apoptosome -regulatory pathway, including the mechanism by which PETCM initiates the pathway, and the mechanism by which PHAP stimulates apoptosome activity. In addition, we will determine the structural basis for the apoptotic and tumor suppressive activities of PHAP, and test whether the apoptotic activity of PHAP contributes to its tumor suppressive property. In conducting these studies, techniques of biochemistry, molecular biology, cell biology, and chemical biology will be used. This researchwill elucidate the molecular mechanisms of the death regulatory pathway and facilitate understanding of its physiological functions. This work should also shed light on designing novel cancer therapies by targeting this pathway.